Field of the Invention
This invention generally concerns the use of derivatives/analogues of poly(vinylpyrrolidone) (PVP), namely poly(pyrrolidone) macrocyclics, oligomers and low molecular weight polymers as masking agents for biological materials in a manner analogous to those obtained using poly(ethyleneglycol) (PEG). Additionally, the small molecule pyrrolidone intermediates (i.e., the monomeric precursors) as well the new poly(pyrrolidone) oligomers/polymers obtained from these derivatives exhibit unique intrinsic fluorescence (IF) or non-traditional fluorescence (NTF) properties that cannot be explained by traditional photochemistry and fluorescence paradigms. These compounds, oligomers and polymers have a variety of applications such as in masking drugs for biological applications, cellular imaging, gene transfection, biosensing, fluorescence directed surgical resections, drug delivery, forensics, mineral/gemstone characterization, oil field enhancement and diagnostics, counterfeit goods detection, tracer studies related to liquid/water flow, fluorescent whitening agents and LED display enhancements and others.
Description of Related Art
PEGylation
The general concept of PEGylation was first introduced and patented in the 1970's by F. Davis et al., (U.S. Pat. No. 4,179,337 (1979)) as a strategy to reduce toxicity, immunogenicity and proteolytic degradation of therapeutic drugs/proteins, while enhancing blood circulation times, drug solubility and PK/PD's of the therapy. The strategy involves the covalent conjugation of poly(ethylene glycol) oligomers (i.e., DP=4-50) to the desired therapeutic drug or substrate. This PEGylation strategy has provided the basis for launching over a dozen important drugs that include: for cancer treatment such as Doxil/Caelyx® by Ortho/Schering-Plough (2001), multiple sclerosis, such as Plegridy® by Biogen (2014), cancer related drug Movantik® by AstraZeneca (2014), anemia such as Peginesatide® by Affmax Takeda Pharma, (2012), to mention a few. This strategy involves the covalent conjugation of low molecular weight PEG oligomers (i.e., <50-70 KDa) using so-called, “activated PEG reagents” as described below:
where: X and Y can be independently reactive or non-reactive with functionality possessed by the desired protein, polynucleotide or therapeutic drug to be modified; n is from 4-50.
POXylation
Due to a number of shortcomings related to PEG oligomer chemical properties (i.e., oxidative, enzymatic stability or immunogentic problems with chronic use and due to higher MW fractions), (G. T. Hermanson, Chapter 18 in Bioconjugate Techniques, Second Ed., (2008) 707-742), there has been an active quest for alternative polymer types and compositions. This has led to an early report by Zalipsky et al. (see S. Zalipsky, et al., J. Pharm. Sci., (1996), 85, 133-137) describing the usefulness and potential advantages of poly(oxazolines) as a replacement for PEG's. Very recently, work has focused on the use of poly(oxazolines) (F. M. Veronese, et al., Bioconjugate Chem., (2011), 22, 976-986) and POXylation conjugates of rotigotine (i.e., dopamine agonist) for the treatment of Parkinson's disease by Serina Therapeutics (www.serinatherapeutics.com). As such, these POXylation protocols involve the use of “activated POX reagents” for attachment to these therapeutic drugs/proteins as described below:
where: X and Y can be independently reactive or non-reactive with functionality possessed by the desired protein, polynucleotide or therapeutic drug—to be modified; n is from 4-50.
Historical Use of Poly(Vinylpyrrolidone) (PVP) as an Injectable Synthetic Polymer in Humans
Historically, the most extensively studied/documented synthetic polymer composition utilized for internal injection in humans has been poly(vinylpyrrolidone) (PVP). For over 75 years, since its discovery in the late 1930's, this polymer has been injected in over 500,000 human patients with virtually no adverse toxicity, immunogenicity or other negative effects for use as a very successful blood substitute/extender (Sultana, et al., J. Pakistan Med. Association, (1978), 28 (10), 147-153). More extensive and contemporary human use of this synthetic polymer composition as an injectable has been hampered solely by concerns that higher molecular weight polymer fractions (i.e., >70 KDa) may not be adequately excreted through the kidney and be accumulated in vivo with multiple injections and over extended time. This hypothesis appears to have been confirmed by several well documented medical studies (Wang et al., J. Cutan Pathol., 2006, 33, 454-457). The specific medical condition created is referred to as; PVP accumulation disease or Dupont-Lachapelle Disease. This medical condition is widely recognized to be due to non-excretable, higher molecular weight PVP fractions present in currently available poly(pyrrolidone) products. Although this medical condition is not fatal, it is considered to be a negative feature for PVP since higher molecular weight PVP fractions have been proven difficult if not impossible to remove (Pfirrmann et al., U.S. Pat. No. 6,080,397, 2000) and will require a significant scientific solution or alternative for future use.
Currently, PVP polymers are produced commercially in large quantities by various manufacturers such as BASF. PVP production involves the free radical polymerization of N-vinyl pyrrolidone monomer (N-VP). The N-VP monomer is obtained by the original Reppe process involving the combination of acetylene, formaldehyde and ammonia under high pressure (see Scheme 1 below).

The (N-VP) monomer has been successfully polymerized only with free radical catalysts (i.e., peroxides, persulfates) in bulk, solution or in suspension, to give linear-PVP with weight-average molar masses ranging from 2500-1,000,000 Da (see Haaf et al. Polymer Journal, 1985, 17(1), 143-152). These linear-PVP polymers are generally obtained as highly poly-dispersed products and were characterized by measuring their viscosities in solution according to a “Fikentscher K-value molecular weight relationship” developed by Kern and Cherdron (Kern et al., Houben Weyl, Methoden der Organische Chemie, Vol. 14, 4th ed., Georg Thieme Verlag, Stuttgart, 1961, p. 1106), using the relationship between Mw, Mn and K; wherein: Mw=15K2/3 and Mn=24K2. The letter K together with an appropriate number is used to relate the molecular weight for the various PVP molecular weight fractions. For example a K-12 has an average molecular weight of ˜20,000 Da and K-90 has a molecular weight of ˜1M Da. Therefore, PVP with a specified K-value and average molecular weight consists of a range of molecular sizes. Based on these viscosity characterization protocols it is clearly apparent that well-defined molecular weight ranges let alone well defined, controllable molecular weights for PVP do not currently exist. Controlling PVP polymer molecular weights, which is related to nanoscale sizes, is a critical issue for many nanomedicine applications, wherein, nanoscale size is known to determine excretion modes, bio-distributions, toxicology and complement activation properties (see for example Kannan et al., J. Intern. Med. 2014, 276, 579-617).
The pyrrolidone moiety as found in PVP (i.e., Povidone, trademark of BASF) enjoys an excellent record and universal recognition as a versatile non-toxic, biocompatible, physiologically inert material for a wide variety of medical applications (see Haaf et al., Polymer Journal, 1985, 17(1), 143-152). Foremost has been the extensive in vivo use of PVP as a blood plasma extender (e.g., Sultana, et al., J. Pakistan Med. Association, 1978, 28, (10), 147-153); wherein, it has undoubtedly saved countless lives. During World War II, (i.e., initiated by I. G. Farben; now Providone by BASF) and in subsequent years (Korean War) [http:/hcvets.com/data/military/korea.htm; page 44], referred to in Sweden as Periston; it is documented that PVP has been used internally via injection in over 500,000 human recipients as a blood extender (Sultana, et al., J. Pakistan Med. Association, 1978, 28, (10), 147-153) without any significant evidence of deleterious effects (Ravin et al., New England J. of Med., 1952, 247, 921-929). Radioactive studies showed that 95-100% of injected PVP (i.e., Periston) was excreted via the urine within 72 hours; 40% was excreted within 20 minutes; and within 6 hours, virtually all circulating PVP had disappeared from the plasma. Subsequent research has shown that the (PVP) composition exhibits virtually no antigenic properties (Maurer et al., J. Immunology, 1956, 77(2) 105-110) compared to other synthetic (i.e., polyesters/polyalcohols) or biological polymers (i.e., poly(dextrans) or poly(saccharides)).
Currently, (PVP) is being used as an adjuvant for immobilizing spermatozoa for in vitro fertilization protocols (www.coopersurgical.com). Other examples include the use of PVP in applications ranging from cosmetics (e.g., hair sprays) to eye drops and oral pill binding formulations. More recent confirmation of the low cytotoxicity and minimal interaction of the poly(pyrrolidone) moiety with proteins, when presented on the surface of poly(amidoamine) (PAMAM) dendrimers has been reported (Ciolkowski et al., Nanomedicine, NBM, 2012, 8, 815-817; and Janaszewska et al., Nanomedicine, NBM, 2013, 9, 461-464).
As such, there is a critical need to synthesize/control the PVP molecular weight in a range of 3-14 KD (Pfirrmann et al., U.S. Pat. No. 6,080,397, 2000). This is necessary to avoid in vivo accumulation of higher MW PVP fractions and be acceptable as an injectable product for in vivo applications. Unfortunately, all known polymerization mechanisms (i.e., free radical, anionic, cationic types) for propagating N-vinyl pyrrolidone to produce PVP polymers lead to substantial amounts of uncontrolled, higher molecular weight (i.e., >14 KDa). PVP products, as well as polymerization side products that make these materials unacceptable for in vivo or injectable product applications. More specifically, when PVP containing higher molecular weight fractions (i.e., >14 KDa) are administered intravenously, an in vivo accumulation of the polymer may occur which is referred to as “PVP storage disease” or also known as the Dupont-Lachapelle Disease (Wang et al., J. Cutan Pathol., 2006, 33, 454-457). This disease is characterized by symptoms that include dermatosis, rheumatic joint pain, and pulmonary respiratory insufficiency. On the other hand, low molecular weight PVP with a molecular weight of <14 KDa and a K-value less than 17 has been found to be non-allergenic and is quickly removed unchanged by excretion from the blood stream via the kidneys. However, all attempts at producing low MW PVP exclusively by free radical polymerization and subsequent ultra-filtration have been unsuccessful (Pfirrmann et al., U.S. Pat. No. 6,080,397, 2000; www.rloginconsulting.com/ . . . pyrrolidone %20backbone %20polymers.pdf).
Fluorescence Discussion
Fluorescence occurs when an orbital electron of an atom, molecule, polymer or nanostructure in the ground state (S0) is excited to a higher quantum state (S1) by the absorption of some form of energy (i.e., usually a photon; hvex) and then relaxes back to the ground state (see FIG. 1). This two-step process is described as:
1. Excitation: (S0)+hvex→(S1)
2. Fluorescence (emission): (S1)→(S0)+hvem+heat
This relaxation or return [i.e., (S1)→(S0)] to the ground state is accompanied by the emission of lower energy photons of light (hvem), which is referred to as fluorescence (see FIG. 1). (The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 2010, 11th Ed.)
A fluorophore in the (S1) state may return to the ground state (S0) via relaxation pathways involving either radiative emission (i.e., fluorescence), non-radiative events (i.e., heat) or internal intersystem conversion (IC) (i.e., intersystem crossover, (ISC)) to a non-fluorescent triplet excited state (T1) with time scales on the order of 10−10 to 10−9 seconds.
These (T1) species are very sensitive to molecular oxygen and may undergo redox reactions leading to highly reactive superoxide radicals (ROS) and irreversible fluorophore damage referred to as “photobleaching” (Q. Zheng, et al., Chem. Soc. Rev., 2014, 43, 1044-1056). These highly reactive oxygen species (ROS) may cause fluorophore degradation or cause phototoxicity by reacting with nearby biomolecules and are in fact pivotal to so-called photodynamic therapies employed in nanomedicine.
Due to vibrational relaxations following excitation, the photon energy emitted from (S1) will generally be lower than the excitation photon. This results in an increase in the fluorescence emission wavelength which may range from 5-50 nm higher than the excitation wavelength. The difference between the excitation wavelength and the emission wavelength is referred to as the Stokes shift (N. J. Turro et al., Modern Molecular Photochemistry of Organic Molecules, 2010 University Science Books, The Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 2010, 11th Ed.).
Fluorescence imaging techniques have broad applications in life sciences and clinical research. However, these techniques critically rely on bright and photostable fluorescent probes. Currently available fluorescent probes for biological imaging mainly include organic fluorophores (Terai and Nagano, Pflugers Archiv. European J. Physiology, 2013, 465, 347-359) and quantum dots (Chen et al., Trends Analytical Chemistry, 2014, 58, 120-129). Small organic dyes suffer from several unwanted properties such as poor solubility, problems with targeting desired cell compartments, rapid irreversible photobleaching, and cell leakage. Inorganic nanoconjugates such as quantum dots are exceptionally bright, photostable, and characterized by narrow emission spectra, but they possess important drawbacks. First of all, they are toxic and that can limit their applications in vivo. Moreover, their intracellular delivery raises problems that make it difficult to follow some biological processes (Jamieson et al., Biomaterials, 2007; 28, 4717-4732).
Fluorescence in the field of biology and nanomedicine has become a major research focus due to their broad applications in cellular imaging, biosensing, fluorescence directed surgical resections (R. Tsien et al., Proc. of the National Acad. of Sci., 2010, 107, 4317-4322) and drug delivery (i.e., theranostics) (S. Lo, et al., Mol. Pharmaceutics, 2013, 10, 793-812; R. Tsien et al., Proc. of the National Acad. of Sci. 2010, 107, 4311-4316).
Applications of fluorescence outside of biology/medicine uses include, but are not limited to: forensics (M. Y. Berezin et al., Chem. Reviews, 2010, 110, 2641-2684); oil field enhancement and diagnostics (M. Amanullah, 2013, SPE164162); counterfeit goods detection (U.S. Pat. No. 8,735,852, issued May 27, 2014; Y. Zhang et al., Dyes Pigm., 2008, 77, 545); tracer studies related to liquid/water flow (WO 2011/030313, A method for detecting an analyte, Indian Inst., of Science, Mar. 17, 2011); fluorescent whitening agents; and LED display enhancements (US Pat. Appl. 20140035960, Apple Inc.) and others.
Traditional standard fluorescing agents are usually described as being members of three major categories:
Category I are organic aromatic conjugated polyenes that include small molecules with low molecular weight (<1000 da) and are derived from these conjugated organic aromatics structures.
Category II consists of fluorescent proteins that usually contain one or more of the three key aromatic moieties such as tryptophan, tyrosine and/or phenylalanines.
Category III consists of inorganic nanoparticles derived from cadmium or lead chalcogenides such as heavy metal sulfides or selenides that must have sizes smaller than a Bohr exciton or radius (2-50 nm). Their fluorescence is determined by their size, but do not exhibit the weakness of photobleaching.
The weaknesses of each traditional fluorophore category are as follows: Category I—lack robustness in the presence of oxygen which leads to rapid fluorophore degradation referred to as photobleaching, as well as photo-toxicity resulting from the generation of the reactive oxygen species (ROS) which may cause cellular damage and potential carcinogenicity; Category II: proteins that may denature, lack robustness in the presence of oxygen which leads to rapid degradation referred to as photobleaching, exhibit immunogenicity; Category III: quantum dots exhibit heavy metal toxicity, blinking fluorescence, lack of solubility for in vivo applications, size must be nanometric and precise (2-50 nm).
The pyrrolidone moiety on the other hand which is a critical component of this invention has an excellent record and international recognition as a versatile non-toxic, biocompatible material for a wide variety of medical applications. Foremost has been the extensive use of poly(vinylpyrrolidone) (PVP) as an in vivo blood plasma extender (Polyvinyl Pyrrolidone as a Plasma Expander—Studies on Its Excretion, Distribution and Metabolism, Herbert A. Ravin, Arnold M. Seligman, M.D., and Jacob Fine, M.D.). Since the World War II, it has been used in over 500,000 human recipients without any evidence of deleterious effects (H. A. Ravin, N. Engl. J. Med., 1952, 247, 921-929).
In another feature, dendritic polymers are known in the art and are discussed extensively in DENDRIMERS, DENDRONS, AND DENDRITIC POLYMERS, Tomalia, D. A., Christensen, J. B. and Boas, U. (2012) Cambridge University Press, New York, N.Y. Dendritic polymers have become recognized as the fourth and most recently reported major class of polymeric architecture (J. Polym. Science, Part A: Polym. Chem. 2002, 40, 2719-2728).
Three major architecture components of dendrimers, namely the cores, interior compositions as well as their surface chemistries can be readily modified. At the present, dozens of diverse cores, nearly 100 different interior compositions and over 1000 different surface moieties have been reported for dendrimers [e.g., DENDRIMERS, DENDRONS, AND DENDRITIC POLYMERS, Tomalia, D. A., Christensen, J. B. and Boas, U. (2012) Cambridge University Press, New York, N.Y.]. In many cases dendrimer surface modifications have been performed to alter, enhance or obtain new emerging properties such as: to modify/reduce dendrimer toxicity, gain enhanced solubilities, reduce dendrimer-protein interactions/immunogenicity (i.e., dendrimer stealthness), for the attachment of drugs, targeting or imaging agents including traditional fluorophores such as fluorescein, Rhodamine red or cyanine dyes. Many of these surface chemistry enhanced dendrimer properties have been shown to be invaluable in a variety of life sciences and nanomedicine applications (e.g., U.S. Pat. No. 5,527,524).
As early as 2001, poly(amidoamine) (PAMAM) dendrimers were reported by Tucker et al., (S. Tucker et al., Applied Spectroscopy, 2001, 55, 679-683) to exhibit intrinsic fluorescence properties that could not be explained mechanistically by any known traditional fluorescence paradigm. This new non-traditional fluorescence (NTF), observed in dendrimers, generally required excitation radiation between 250-400 nm, followed by relaxation to the ground state to produce characteristic emission bands that ranged from the visible to near infrared region (i.e., 400-750 nm).
Since this early report, the NTF phenomena has been observed in a wide range of different dendrimer families (i.e., interior compositions) all of which appear to have one thing in common, namely they possess multiples of tertiary amines (3°-amines) and/or amides in their interior backbone compositions. It is notable that dendrimer terminal/surface functionality did not appear to significantly influence (NTF); however, dendrimer generation level (G. Jayamurugan et al., Org. Lett., 2008, 10, 9-12), degree of dendrimer aggregation (P. K. Anthaijanam et al., J. Photochemistry & Photobiology A: Chem., 2009, 203, 50-55), solvent viscosities (P. K. Anthaijanam et al., J. Photochemistry & Photobiology A: Chem., 2009, 203, 50-55), low pH's (T. Imae et al., J. Am. Chem. Soc., 2004, 126, 13204-13205; L. Pastor-Perez et al., Macromol Rapid Commun., 2007, 28, 1404-1409; Y. Wang et al., J. Nanosci. Nanotechnol., 2010, 10, 4227-4233; Y. Shen et al., Chem. Eur. J., 2011, 17, 5319-5326), aging (D. Wang et al., J. Colloid & Interface Science, 2007, 306, 222-227), exposure to air or oxidizing reagents (A. J. Bard et al., J. Am. Chem. Soc., 2004, 126, 8358-8359; T. Imae et al., Colloids & Surfaces B: Biointerfaces, 2011, 83, 58-60), and even a few others, did cause enhancements in fluorescence intensities. In addition to the early more ordered, monodispersed dendrimer examples, the (NTF) phenomena was subsequently observed in several other major macromolecular architectures including: (a) random hyperbranched (Y. Chen et al., Bioconjugate Chem., 2011, 22, 1162-1170), (b) linear (L. Pastor-Perez et al., Macromol Rapid Commun., 2007, 28, 1404-1409) and (c) certain simple branched (S.-W. Kuo et al., J. of Nanomaterials, 2012, 749732, 10 Pages) polymer structures. In spite of many attempts to utilize these unique dendrimer (NTF) properties for imaging biological cells or labeling, the low (NTF) fluorescence emission intensities generally precluded their practical use, except in the presence of certain oxidizing reagents/environment or at low pH's (i.e., 2-3) in order to obtain an adequate emission intensity for certain applications such as gene transfection (Y. Chen et al., Bioconjugate Chem., 2011, 22, 1162-1170).
Applying dendrimers—versatile, globular, monodisperse polymers with many surface functional groups—seems to be a solution that may help to overcome limitations of both single organic fluorophores and inorganic nanoprobes. The size of dendrimers places them on the same scale as fluorescent proteins: they are larger than organic dyes and smaller than quantum dots.
Dendrimers have been used as scaffolds for fluorophores. G2 PAMAM dendrimers with PEG chains have been functionalized with two types of fluorophores: carboxyfluorescein and tetramethyl-rhodamine and tested in Chinese hamster ovary cells (Albertazzi et al., PloS ONE, 2011, 6, e28450. doi:10.1371/journal.pone.0028450). Higher generations G5 and G6 PAMAM dendrimers have been conjugated with multiple cyanine dyes (Kim et al., Biophys. J., 2013, 104, 1566-1575). In many cases covalent attachment of fluorescent labels on the surface of the dendrimer is necessary to evaluate its biological functions in vitro or in vivo. However, such a modification creates a risk of decreased dendrimer biocompatibility, and affects its biodistribution properties. That is why seeking intrinsically fluorescent dendrimers are of paramount importance.
Clearly, having biocompatible compounds that display fluorescence in the desired wavelength and intensity, with low toxicity, for the intended use has commercial application.